The losses due to rotting caused by fungal, and in some cases bacterial pathogens in plant tissues and, more particularly, in fruit and vegetable products, are an important problem on farms.
There are many ways by which fungal pathogens cross the external barriers of plant products and gain access to nutrient-rich tissues that allow them to cause infections. From among them, undoubtedly, the most common entryway is through wounds made both before harvest, and during harvest and during the processing and storage following the same (mechanical damage, damage caused by cold or heat, insect bites, etc.) although some fungi are capable of penetrating through natural openings (stomas, lenticels, peduncle, etc.) and some even without the need of a physical opening on the surface, by means of the direct attack thereof with enzymes and specialized organs which end up causing an opening on the surface (S. Bautista-Baños, 2014. Postharvest Decay: Control Strategies). In either case, one way or another, there is always an access opening on the surface of the plant product where the pathogen enters, allowing it to gain access to the nutrient-rich tissues under the epidemlis, and cause the infection. As a consequence, targeting any fungicide against said openings where the infection begins or may begin (sites of infection) could be enough to achieve efficacy against these pathologies.
Nowadays, however, fungicides are applied as an aqueous solution or emulsion directly bathing or showering the whole fruits, thus covering not only the sites of infection, but also the whole fruit with fungicide, finally leaving the whole fruit with fungicide residues. With these treatments, considerable amounts of synthetic fungicides (potentially toxic for humans and the environment) are unnecessarily introduced into the food chain, and therefore the authorities establish an MRL (maximum residues level) for each fungicide in each fruit and vegetable product in order to regulate this problem. These MRL are the cause of a continuous controversy and update and are frequently deemed as insufficient by NGOs and supermarkets, which further restrict the acceptable residue level for the sale of their products.
In order to reduce the amount of fungicide incorporated into fruits, an interesting approach would be to target and release fungicide only to the site of infection, that is, the site where the infection begins and, thus, the rotting of the fruit.
If only the sites of infection of the plant tissue were treated by means of a selective and controlled treatment, targeted to said sites of infection, the amount of fungicide incorporated to the fruits would be radically lower, since, for example, in manufacturing warehouses, the fruits with small wounds, some of them almost imperceptible to the human eye, are precisely the ones passing the sorting line and reaching the consumer. These small wounds account for a tiny part of the surface of the fruit, such that the introduction of fungicide only inside them may be a breakthrough since the residue of fungicide on the whole fruit could be almost undetectable, that is, it would be found at trace level. The fruits with very large wounds, on the other hand, are easily detected in a sorting line and never reach the consumer.
Many examples of controlled or sustained release of active materials applied to diverse fields, being pesticides among them, have been described. These are systems which release the active ingredient non-specifically to the medium as a function of time, following a certain kinetic, from the moment in which the product is incorporated into said medium.
From, among the different approaches aiming to achieve this object, it is worth noting the use of mesoporous silica nanoparticles or microparticles, with or without functionalization, in which the active is retained in the pores and is progressively released to the medium (S. Jambhrunkar et al., J. Colloid Interf. Sci. 2014, 434, 218-225; A. Janatova et al., Ind. Crop. Prod. 2015, 67, 216-220; G. Q. Lu et al., WO2011054046A1; L. B. Yan and C. A. Martin, WO2011061787A2; D. H. Traynor and H. G. Traynor, US20120074603A1; K. Qian et al., Micropor. Mesopor. Mat. 2013, 169, 1-6; H. Wanyika, J. Nanopart. Res. 2013, 15:1831, 1-9; J. Chen et al., J. Agric. Food Chem. 2011, 59, 307-311; M. Otsuka et al., J. Control. Release 2000, 67, 369-384; Kortesuo et al., J. Control. Release 2001, 76, 227-238). This approach is especially useful for applications in which it is interesting to have a product “reservoir”, when it is interesting to progressively apply the product or when it is interesting to protect the product from the medium. However, it is not very convenient for treating selectively the sites of infection with fungicides, as it implies the use of unspecific systems. Thus, processing waters would accumulate fungicides over time depending on the kinetics of the product and would be polluted with residues. Likewise, these fungicides in water would pass into fruits unspecifically, leaving residues all over the surface. On the other hand, the release shall not be progressive, as it is best to have all the dose available at the moment of treatment in order to eradicate the infection within the permissible delay time between inoculation and treatment. Otherwise, the treatment loses its effectiveness (B. L. Wild and L. J. Spohr, Aust. J. Exp. Agr. 1989, 29, 139-142).
On the other hand, there are many examples described about selective and targeted release, also applied to very diverse fields, wherein the release is zero until a stimulus triggers the release of the active material. In this approach, it is worth mentioning the systems based on mesoporous silica gel particles wherein the pores loaded with active material are closed with molecular gates sensitive to specific stimuli. When the stimulus occurs, the gates are open and the active is released (C. Coll et al., Accounts Chem. Res. 2013, 46, 339-349). The stimulus capable of opening said molecular gates may be the presence of an enzyme capable of degradating the molecules acting as gates (A. Bernardos et al., Angew. Chem. Int. Ed. 2009, 48, 5884-5887; N. Mas et al., Chem. Eur. J. 2013, 19, 1346-1356; C. Coll et al., Angew. Chem. Int. Ed. 2011, 50, 2138-2140; A. Agostini et al., Anger. Chem. Int. Ed. 2012, 51, 10556-10560; A. Agostini et al., Langmuir 2012, 28, 14766-14776; A. Bernardos et al., ACS Nano 2010, 4, 6353-6368), the presence of a specific molecule capable of interacting with the gates and opening them (M. Chen et al., Chem. Commun. 2012, 48, 9522-9524; E. Climent et al., J. Am. Chem. Soc. 2009, 131, 14075-14080; R. Casasus et al., J. Am. Chem. Soc. 2008, 130, 1903-1917; A Bernardos et al., J. Control. Release 2008, 131, 181-189; Q. Yang et al., Chem. Mater. 2005, 17, 5999-6003; R. Casasus et al., J. Am. Chem. Soc. 2004, 126, 8612-8613), a change in pH (R. Casasus et al., J. Am. Chem. Soc. 2008, 130, 1903-1917; A. Bernardos et al., J. Control. Release 2008, 131, 181-189; R. Casasus et al., J. Am. Chem. Soc. 2004, 126, 8612-8613) or a change in temperature (E. Aznar et al., Angew. Chem. Int. Ed. 2011, 50, 11172-11175), among others.
This application may be adequate in principle for the selective treatment of sites of infection, such as for example wounds, with fungicides, since the release would be theoretically zero in processing waters and also on the surface of the fruit and the fungicide would be released only as a consequence of a stimulus capable of opening the molecular gates produced by the wound or by the fungus itself inoculated at the site of infection. One of the most feasible options would be producing an enzymatic opening caused by any hydrolytic enzyme produced by the fungus itself inoculated at the site of infection. However, the gap generated between the onset of the infection and the moment when the complete dose is reached by the opening of the gates could make the treatment ineffective, since the opening of said gates would be precisely the consequence of the growth of the fungus. That is, when the growing fungus produces sufficient enzyme to open enough gates and deliver a good treatment dose, it will be already in an advanced stage of growth and it would be probably too late to eradicate it.
For treatment to be selective and also effective, the product should be capable of maintaining a zero-fungicide release in the treatment water and on the surface of the fruit and at the same time releasing said fungicide instantaneously on the site of infection, without time gaps, as the effectiveness of the same is highly dependent of the time elapsed between inoculation and treatment, which in turn corresponds to the time of advance of the infection.
The present invention overcomes this problem through a system able to hold and release the fungicide only selectively at the site of infection, but at the same time being totally open, without gates, for the free and immediate release of the whole fungicide dose at the site of infection.